Phycobiliproteins are a family of colored proteins which serve as components of the light-harvesting apparatus of a variety of cyanobacteria and red algae. In vivo the phycobiliproteins occur as macromolecular assemblages, known as phycobilisomes, attached to thylakoid membranes. The structure and comparative biochemistry of phycobilisomes and phycobiliproteins have been the subject of numerous reviews (see for example: Glazer, A. N., Ann. Rev. of Microbiol 36:173-98 (1982) and Glazer, A. N., Biochem. Biophys. Acta 768: 29-51 (1984). Briefly, phycobiliproteins (or as they are also known, biliproteins) possess a basic monomer structure comprised of two dissimilar polypeptides (i.e. an .alpha.,.beta. subunit structure). A third subunit (.gamma.) as well as various other linker polypeptides may also be associated with the basic monomer or multiples thereof. The characteristic absorbance of the various biliproteins is due in large measure to the existence of open-chain tetrapyrrole prosthetic groups which are covalently attached to the .alpha., .beta. or .gamma. subunits by means of at least one thioether bond. A number of factors dictate the particular spectroscopic properties that individual types of biliproteins will display, these include the number and type of prosthetic groups, the conformational relationship between the prosthetic groups and the subunit to which they are attached and changes in the structural environment due to the formation of molecules of higher aggregation states.
FIG. 1 illustrates the number and types of bilin prosthetic groups among the subunit of various biliproteins and Table 1 summarizes some properties of common phycobiliproteins.
TABLE 1 ______________________________________ PROPERTIES OF PHYCOBILIPROTEINS Dis-tri-bu- Absorptionmaxima inthe visible.sup.2 Fluores-cence emis-sion maxi- ##STR1## Biliprotein tion.sup.1 (nm) mum.sup.2 (nm) Ratio ______________________________________ Allophycocyanin B C,R 671 618 680 -- Allophycocyanin C,R 650 660 -- C-Phycocyanin C,R 620 637 -- R-Phycocyanin R 617 555 63 -- Phycoerythrocyanin C 568 590(s) 625 -- C-Phycoerythrin C 565 577 -- b-Phycoerythrin R 545 563(s) 570 -- B-Phycoerythrin R 545 563 498(s) 575 0.06 R-Phycoerythrin C,R 567 538 498 578 0.36 ______________________________________ .sup.1 C = cyanobacteria; R = red algae. .sup.2 For a given biliprotein, the exact positions of the absorption and emission maxima vary somewhat depending on the organism that serves as th source of the protein and on the method of purification.
As can be seen from Table 1, red algae possess phycoerythrins displaying visible absorption spectra with peaks at about 566 nm and peaks or shoulders at about 540 and 500 nm with varying relative intensities. Red algal phycoerythrins carry two types of covalently attached tetrapyrrole prosthetic groups, phycoerythrobilin (PEB) and phycourobilin (PUB). The PEB groups give rise to the 566- and 540-nm peaks, and the PUBs give rise to the 500-nm peak. In contrast, phycoerythrins purified from cyanobacteria isolated from soil or fresh water contain only PEB groups and do not exhibit the 500-nm peak. The cyanobacterium Gloeobacter violaceus does contain a phycoerythrin with both PEB and PUB chromophores, but the organism is atypical in other respects as well (Bryant, D. A. et al., Arch Microbiol. 129:190 (1981) and Rippka R, et al, Arch. Microbiol. 100-419 (1974). The difference in the bilin composition of red algal and cyanobacterial phycoerythrins may be related to the changing nature of solar radiation as it penetrates seawater. Marine algae are exposed to maximum transmission of light at approximately 500 nm and the presence in these organisms of a photosynthetic accessory pigment (PUB) that absorbs maximally at this wavelength appears to be more than coincidental.
Unicellular cyanobacteria containing phycoerythrin have been observed in abundance among marine phytoplankton (Waterbuty, J. B., et al, Nature 277:293 (1977)), and these organisms make a major contribution to the primary productivity in the ocean. Although the phycoerythrin of several of these organisms is of the ordinary cyanobacterial type (C-phycoerythrin), it is interesting that many strains contain phycoerythrins with both PEB and PUB chromophores (Fujita, Y. and S. Shimura, Plant Cell Physiol 15:9393 (1974) Alberte et al., (Plant Physiol. 74: 732-737 (1984). Kusar et al. Pro. Nat'l. Acad. Sci. USA 78: 6888-6897 (1981)). Phycoerythrin purified from a marine Synechococcus contains the highest content of PUB of any known phycoerythrin (Ong, et al. Science 224: 80-83, (1984).
In a recent survey of marine Synechococcus spp. Alberte et al, (Plant Physiol 74: 732-739 (1984) identified two apparently novel types of Synechococcus based upon their spectral and biochemical properties. The first type (Type II-PE) possesses a phycoerythrin exhibiting a single broad absorption maximum at 551 nm and a fluorescence emission peak at 570 nm. The second type (Type I-PE) is characterized by phycoerythrins exhibiting adsorption maxima at 558 and at 500 nm and a fluorescence emission peak at 560 nm. When absorption measurements are made after acid-urea treatment Type II-PE displays an absorption maximum at 558 nm while the Type I displays maxima at 558 and 500. The absorption at 558 is presumably due to phycoerythrobilin (PEB) however the "classic" PEB as present in the C-, R- and b- PEB exhibit at maximum absorbance at 550 nm in acid urea. The shift of 8 nm to red is unexplained and the authors refer to this species as "PEB-like". The absorption maximum at 500 nm in the Type I-PE is ascribed to the phycourobilin (PUB) chromophore. The ratio of PUB/PEB-like chromophores in Type I-PE is 1.3/4.9=0.26.
The utility of phycobiliproteins as components of reagents for fluorescence analysis of molecules and cells has been described by Oi, V. T. et al., (J. Cell. Biol. 93:981 (1983)) by Stryer et al., (Eur. Pat. Appln. 0.076694, Apr. 4, 1983) and by Kronick, M. N. and Grossman, P. D., (Clin. Chem. 29 (9): 1582 (1983)). Oi, et al. and Stryer et al. disclose the use of phycobiliprotein conjugated to immunoglobulins, protein A, biotin and avidin and the application of these conjugates to fluorescence-activated cell sorting, fluorescence microscopy, and fluorescence immunoassays. R-phycoerythrin (from Gastroclonium coulteir), C-phycocyanin (from Synechococcus), allophycocyanin (from Anabaena variabilis) and B-phycoerythrin (from Porphyridium cruentum) were employed in forming the conjugates. Kronick and Grossman (supra) disclose the use of B-phycoerythrin (from P. cruentum) coupled to rabbit anti-human IgG in solid phase "sandwich-type" immunoassays. These references to the extent they provide the background necessary for the skilled artisan to practice this invention are incorporated herein by reference.
This invention is predicated upon the discovery of a unique new type of phycoerythrin exhibiting particular advantageous spectral properties and that inter alia under appropriately selected conditions the sensitivity of assays employing this phycoerythrin is nearly doubled.